Design, synthesis, in vitro anticancer, molecular docking and SAR studies of new series of pyrrolo[2,3-d]pyrimidine derivatives

The current study involves the design and synthesis of a newly synthesized pyrrolo[2,3-d]pyrimidine derivatives to contain chlorine atoms in positions 4 and 6 and trichloromethyl group in position 2 using microwave technique as a new and robust approach for preparation of this type of pyrrolo[2,3-d]pyrimidine derivatives. The chemical structure of the synthesized pyrrolo[2,3-d]pyrimidine derivatives 3–19 was well-characterized using spectral and elemental analyses as well as single-crystal X-ray diffraction. All compounds were tested in vitro against seven selected human cancer cell lines, namely, MCF7, A549, HCT116, PC3, HePG2, PACA2 and BJ1 using MTT assay. It was found that compounds 14a, 16b and 18b were the most active toward MCF7 with IC50 (1.7, 5.7, and 3.4 μg/ml, respectively) relative to doxorubicin (Dox.) (26.1 μg/ml). Additionally, compound 17 exerted promising cytotoxic effects against HePG2 and PACA2 with IC50 (8.7 and 6.4 μg/ml, respectively) relative to Dox. (21.6 and 28.3 μg/ml, respectively). The molecular docking study confirmed our ELISA result which showed the promising binding affinities of compounds 14a and 17 against Bcl2 anti-apoptotic protein. At the gene expression level, P53, BAX, DR4 and DR5 were up-regulated, while Bcl2, Il-8, and CDK4 were down-regulated in 14a, 14b and 18b treated MCF7 cells. At the protein level, compound 14b increased the activity of Caspase 8 and BAX (18.263 and 14.25 pg/ml) relative to Dox. (3.99 and 4.92 pg/ml, respectively), while the activity of Bcl2 was greatly decreased in 14a treated MCF7 (2.4 pg/ml) compared with Dox. (14.37 pg/ml). Compounds 14a and 14b caused cell cycle arrest at the G1/S phase in MCF7. Compounds 16b and 18b induced the apoptotic death of MCF7 cells. In addition, the percentage of fragmented DNA was increased significantly in 14a treated MCF7 cells. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-023-01014-0.


Introduction
Cancer or tumor cells are known as abnormal growth of cells in the body.According to ACS (American Cancer Society), cancer was estimated to cause 1700 deaths daily and causes about 15% of all human deaths worldwide [1,2].As reported in the GLOBOCAN 2020, female breast cancer (with around 2.3 million new cases, 11.7%) surpassed lung tumor (11.4%) as the most commonly diagnosed cancer, followed by colorectal (10.0%), prostate (7.3%) [3].By 2030, it is predicted that pancreatic cancer will be in the second stand of cancerrelated death after lung tumors [4].
Apoptosis is a cellular death mechanism that plays a critical role in both physiological and pathological conditions.It has a great impact on cellular development and homeostasis [5].It serves to remove any unwanted cells and is a highly regulated mechanism.DNA damage or uncontrolled proliferation triggers the activation of the apoptotic pathway [5].Both intracellular and extracellular signals activate the apoptotic pathway.According to the type of signal, apoptosis divides into two pathways (intrinsic and extrinsic).They are also called the mitochondrial and death receptor pathways, respectively.The intracellular signals involve growth factor deficiency; cytokine deficiency and DNA damage [6], while the extracellular signals are death signals induced by cytotoxic T cells in response to infected or damaged cells [6].Caspases (cysteine aspartyl-specific proteases) are a family of cysteine proteins that degrade specific proteins resulting in apoptosis [7].The caspases are classified into four initiators (caspase-2, -8, -9, -10) and three executioners (caspase-3, -6, -7) [7].The role of the executioner caspases is to degrade the target proteins leading to cell death.Apoptosis evasion, angiogenesis and uncontrolled growth are the most common hallmarks of cancer that are present in all cancer cell types [8].One of the main functions of apoptosis is to prevent cancer [8].The control of restoring or terminating uncontrolled growth by using the apoptotic process is a highly effective cancer treatment method.Therefore, targeting apoptosis is effective for all cancer types.Many anticancer drugs target various stages in both the intrinsic and extrinsic pathways [5].
The use of chemotherapy in the treatment of tumors has opened new possibilities for improving the quality of life of cancer patients and for the cure of disease.Chemotherapeutic drugs are cytotoxic and can kill both normal and malignant cell types.Their usefulness in the treatment of malignancy relies on the assumption that cancer cells, which are rapidly proliferating cells, take up extracellular materials at a higher rate than normal cells [9,10].
Due to the ability of the nitrogen-containing heterocycles to create hydrogen bonding, van der Waals forces, hydrophobic effects, π-stacking interactions and dipole-dipole interactions with the biological targets, there more than 80% of the marketed drugs approved by the FDA [11][12][13][14][15].The pyrrolopyrimidine derivatives are nitrogen-fused heterocycles in which they commonly display a wide array of biological and pharmacological properties and they are found in various small molecule drug design programs [16].
In continuation with our research studies focusing on the synthesis of novel nitrogen-containing heterocycles with anti-cancer activity [12,13,[28][29][30][31][32].Recently, we have been attentive in carrying out the preparation of new pyrrolo [2,3-d]pyrimidine derivatives, with expected biological activity as anti-BVDV (Bovine Viral Diarrhea Virus), under environmentally friendly, timesaving microwave-assisted conditions.Accordingly, we reported that the presence of the trichloromethyl group at the 2-position and chlorine atom at the 6-position of the pyrrolo [2,3-d]pyrimidine scaffold increased the antiviral effect on Bovine Viral Diarrhea Virus (BVDV) [20].Consequently, as depicted in Fig. 1, we used our robust method using microwave-assisted conditions to prepare a new series of pyrrolo [2,3-d]pyrimidine derivatives having the trichloromethyl group on carbon 2 and chlorine atom on carbon 6 with different substitutions on carbon 4 to evaluate the anti-cancer activity of these compounds as a first study for this type of pyrrolo [2,3-d]pyrimidines as anti-cancer agents.Our target is to study the mechanism of action of our compounds as anticancer agents through studying the effect on cell cycle and apoptosis at the gene, protein and DNA level using RT-PCR, Eliza and DNA fragmentation assays, respectively.
In an effort made to find a new pyrrolo [2,3-d]pyrimidine derivatives with promising anti-cancer activities, twenty-five derivatives were synthesized in this study and their relative anti-cancer activities were investigated in an in vitro study.In addition, the molecular docking study and the structure-activity-relationship (SAR) were presented and discussed.

Chemistry
Digital Gallen Kamp MFB-595 instrument was used for recording the melting points for all the compounds and may be uncorrected.The chemicals of 4-Bromoacetophenone, 3,4-Dimethoxyacetophenone, hydrazine monohydrate, malononitrile, formic acid, POCl 3 , pyrrolidine, morpholine, and N-methyl piperazine were used as received without prior purification.IR spectra (cm −1 ) were recorded on a JASCO spectrophotometer using a KBr pellet.The 1 H-NMR and 13 C-NMR spectra were recorded on Bruker spectrometer (400 MHz and 100 MHz, respectively) in deuterated dimethyl sulfoxide (DMSO-d 6 ). 1 H-NMR spectra were assigned relative to deuterated solvent signals, while they reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), and coupling constant (J in Hz).Elemental analyses were recorded in the micro-analysis center at Cairo University.The TLC technique was used to check the purity of the newly synthesized compounds [20].

General procedure for preparation of 4a and 4b
A mixture of pyrrolo [2,3-d]pyrimidin-4-one derivatives (3a or 3b) and the required equivalent number of P 2 O 5 was added in twice as much equivalents of POCl 3 .The mixture was heated in the microwave oven irradiation with 800 W at 100 °C for 15 min.After cooling, the mixture was poured onto ice water and alkalinized with a saturated potassium carbonate solution.The precipitate was filtered off and recrystallized from ethanol to afford 4a or 4b as follows: [20]

Single crystal X-ray diffraction
Single Crystals suitable for X-ray diffraction analysis were obtained from a saturated acetonitrile solution at room temperature.The X-ray crystal structures were determined by using a Rigaku R-AXISRAPID diffractometer and Bruker X8 Prospector.The collection of single crystal data was made at room temperature by using Cu-Kα radiation.The structures were solved by using direct methods and expanded using Fourier techniques.Thenon-hydrogen atoms were refined anisotropically [20,34].
The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group C 2/c, as monoclinic with Z = 8 for the formula unit, C 20 H 13 Cl 4 N 3 O 2 and the formula weight 469.13.The final anisotropic full-matrix least-squares refinement on F2 with 200 variables converged at R1 0.0959%, for the observed data and wR2 = 0.2776% for all data.The goodness-off was 1.059.The largest peak in the final difference electron density synthesis was 2.084 e − /A3 and the largest hole was − 0.743 e−/A3 with an RMS deviation of 0.098 e−/A3.Based on the final model, the calculated density was 1.473 g/cm3 and F(000), 1904 e−.The CCDC number is 2215014.More details could be found in Additional file 1.

Anticancer activity MTT assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to evaluate the percentage of the viability of cells under the effect of the synthesized compounds [35][36][37].All of the following steps were done in a sterile Laminar flow class II biosafety cabinet (Baker, SG406INT, Sanford, ME, USA).DMEM was used as a suspension medium for breast cancer (MCF7), colorectal cancer (HCT116), prostate cancer (PC3), liver cancer (HePG2), pancreatic cancer (PACA2) and lung cancer (A549), while normal cell line (BJ1) was suspended in DMEM-F12 medium.The selected cell lines were purchased from American Type Culture Collection (ATCC).The media were supplemented with a 1% antimycoticantibiotic mixture (10,000 µg/ml streptomycin sulfate, 25 µg/ml Amphotericin B, and 10,000 U/ml Potassium Penicillin) and 1% l-glutamine.10 × 10 4 cells/well were seeded in 96 well microtiter plates and incubated for 24 h at 37 ℃ under 5% carbon dioxide using a water-jacketed Carbon dioxide incubator (Sheldon, SCO5A, OR, USA).Then, the media were discarded and replaced with a fresh one (without serum), and the cells were incubated either alone (negative control) or with different concentrations of the prepared compounds to give a final concentration of (100-50-25-12.5-6.25-3.125-1.56 and 0.78 µg/ ml).After the incubation time of 72 h, the medium was removed.Afterward, 40 µl MTT salt (2.5 μg/ml) was added to each well and kept for 4 h under the same conditions of 37 ºC and 5% carbon dioxide.Finally, 200 μl of 10% Sodium dodecyl sulfate (SDS) in deionized water was added to each well and incubated overnight at 37 ºC.The step of SDS addition was done to stop the reaction and dissolve the formed formazan crystals.The absorbance was then measured at a wavelength of 595 nm and the reference wavelength was set at 620 nm using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3356, California, USA).Doxorubicin was used as a positive control that gave 100% lethality at a concentration of 100 µg/ml under the same conditions.A statistical significance analysis was determined between the samples and the negative control using an independent t-test by SPSS 11 program.Dimethyl sulfoxide (DMSO) was the vehicle that dissolved our synthesized compounds and its concentration in the cells was less than 0.2%.IC 50 values were calculated using the GraphPad Prism 6 program.The degree of selectivity of the synthetic compounds was expressed as SI = IC 50 of a pure compound in a normal cell line/IC 50 of the same pure compound in the cancer cell line, where IC 50 is the concentration of the compound required to kill 50% of the cells.

Molecular docking study
Molecular modeling studies for compounds 14a and 17 were done according to Fathi et al. [38] using (MOE) program 2009.10 version to investigate the ligand-protein interactions at the active sites of the P53 mutant Y220C and Bcl2 proteins.The x-ray crystallographic structures of selected proteins were downloaded from the protein data base (PDB) (www.rcsb.org) (PDB ID: 5O1H and 6QGG respectively).The selected proteins were first prepared for modeling study where the standard ligand molecule was removed from the active site of the protein, the heavy atoms were kept fixed, and the hydrogen atoms were added to the whole protein structure.According to the author's instructions, the RMS gradient was adjusted at 0.01 kcal/mole, the RMS distance at 0.1 Å, and the partial charges were computed using the MMFF94x force field.At the final step, the ligand interaction (MOE) structure was saved as a Pdb file which was then visualized through the BIOVIA Discovery Studio V6.1.0.15350 program, where the tested compounds appeared to fit into the active domain of proteins in 2D and 3D states [39].

Isolation of total RNA and RT-PCR
All of the extractions were conducted on ice with icecold reagents.Total RNA from the different cell lines was isolated using Trizol (Invitrogen; Life Technologies, USA) according to the method of Sthoeger et al. [40].The 260:280 ratios were measured to determine RNA quality after the isolation method was completed according to the manufacturer's instructions.A high-capacity cDNA reverse transcription kit was used to produce complementary DNA (cDNA) (Applied Biosystems, USA).Table 1 shows the primers that were used in these tests.The relative gene expression method (i.e., ΔΔCT) was used to analyze the real-time PCR data, as explained in Applied Biosystem User Bulletin No. 2. Each sample and gene were normalized using the β-actin gene.

ELISA
Elisa assay was used to determine the concentrations of Human caspases-3, -8, Bax, and Bcl2 in PACA2-and A549-treated cells.Also, the concentrations of caspase-8, Bax, and Bcl2 in MCF7-treated cells were detected [41][42][43][44].The procedures were followed up according to the instructions described in the following kits; Invitrogen human Caspase-3 Elisa Kit, Catalog KHO1091, DRG ® human Caspase-8 ELISA Kit, Catalog (EIA-4863), Human Bax ELISA Kit (ab199080) and Human Bcl-2 ELISA Kit (ab119506) respectively.Shortly, the protocol described in the above kits was as followed; at the beginning, all the reagents, samples and standards were prepared.100 μl of samples or standard were micro pipetted to each well of micro-well strip and kept at room temperature for 2 h.After washing the micro-well strips three times with washing buffer, the prepared antibody (anti-rabbit-IgG-HRP) at a concentration of 100 μl was added to each well at room temperature for 1 h.Then, the micro-well strips were washed again three times.Afterward, the prepared tetramethyl-benzidine (TMB) solution (100 μl) was added to the micro-well strips and kept for 10 min.At last, about 50 μl of stop solution was pipetted to each well to inactivate the enzyme completely.The absorbance of all samples was measured at a wavelength of 460 nm and compared to that of the standard.Curve fitting software was used to plot a standard curve and the concentrations for the unknown samples were measured from the standard curve.

Flow cytometer analysis of cell cycle and apoptosis
For cell cycle analysis, 10 6 MCF7 cells were cultured in 60 mm Petri dishes for 24 h and then treated with the selected compounds 14a and 14b at their IC 50 concentrations for 24 h.Herein, the negative control was the untreated MCF7 cells and Doxorubicin was used as a positive control.Firstly, after the treatment time of 24 h, MCF7 cells were centrifuged at 1000 rpm for 5 min at 4 °C.The supernatant was discarded and the cells pellet were washed in phosphate-buffered saline (PBS).Then, the cells pellet were centrifuged at 1000 rpm for another 5 min.Afterward, the cells were collected in a single-cell suspension and maintained in 70% ethanol overnight on ice.Then, cells were washed with 1 ml PBS.At last, 200 µl 1 × propidium iodide (PI) mixture was added to the cells pellet in the dark at room temperature for 30 min.Then, the DNA content was analyzed by subjecting the cells to an Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL).The distribution of cells at different phases of the cell cycle was determined by Multi-cycle software (Phoenix Flow Systems, San Diego, CA).The percentage of apoptotic cells was detected by using Annexin V-FITC kit catalog number (#K101-25).About 2X10 6 of MCF7 and Paca2 cells were washed in 500 µl of 1 × PBS.Then, the cells were centrifuged and the supernatant was discarded.The cells pellet were re-suspended in the annexin V incubation reagent.The annexin V incubation reagent contained 10 μl binding buffer (10 ×), 10 μl propidium iodide, 1 μl annexin V-FITC, and 79 μl deionized water.The cells were incubated in 100 μl annexin V incubation reagent for 15 min in the dark at room temperature [45].Finally, the apoptotic cell percentage was detected by flow cytometry using the FITC signal detector (usually FL-1) and PI staining by the phycoerythrin emission signal detector (usually FL-2).

DNA fragmentation assay in a pancreatic cell line (Paca2), breast cancer cell line (MCF-7) and lung cell line (A549) DNA gel electrophoresis laddering assay
The DNA fragmentation assay in a pancreatic cell line (Paca2), breast cancer cell lines (MCF-7) and lung cell line (A549) was performed in concordance with the premises established by Yawata [46] with some modifications.Briefly, after 24 h of exposure of Paca2, MCF-7 and A549 cancer cell lines to the prepared compounds in different Petri dishes (60 × 15 mm, Greiner), the cells were suspended and homogenized in 1 ml of medium and then centrifuged for 10 min at 900 rpm.The genomic DNA was extracted as shown in Yawata [46].About 1 × 10 6 cells of each tested cancer cell line were treated with the IC 50 values of the tested compounds.All the cells were collected via trypsinization and washed with Dulbecco`s Phosphate Buffered Saline.Then, the cells were treated with the lysis buffer containing 5 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris (pH 7.4), 0.5% Triton X-100 and 150 mM NaCl for 35 min on ice.After that, the lysates were vortexed and centrifuged for 20 min at 10.000 xg.The fragmented DNA was extracted from the supernatant with an equal volume of isoamyl alcohol: chloroform: neutral phenol mixture (1:24:25).Finally, the percentage of fragmented DNA was determined by performing gel electrophoresis using 2% agarose gel containing 0.1 μg/ml ethidium bromide.

Diphenylamine reaction procedure
Pancreatic cancer cell line (Paca2), breast cancer cell line (MCF-7) and lung cancer cell line (A549) were used to determine the percentage of DNA fragmentation after the treatment with IC 50 of the tested compounds.The selected cells were suspended in 0.5 ml of lysis buffer containing 1 mM EDTA, 10 mM Tris-HCl (pH 8) and 0.2% triton X-100.Then, the cells were centrifuged at 10.000 rpm for 20 min at 4 °C.After removing the supernatant, the pellets were re-suspended in 0.5 ml of lysis buffer.Then, 0.5 ml of 25% tri-chloroacetic acid (TCA) was added to the pellets (P) and the supernatants (S), and incubated for 24 h at 4 °C.Afterwards, the cells were centrifuged at 4 °C for 25 min at 10,000 rpm and the pellets were suspended in 80 ml of 5% TCA, followed by incubation at 80 °C for 20 min.Finally, 160 ml of Diphenyl Amine (DPA) solution [150 ml of sulfuric acid, 150 mg DPA in 10 ml glacial acetic acid, and 50 ml acetaldehyde (16 mg/ml)] was added to each cell sample and kept at room temperature for 24 h [47].The percentage of fragmented DNA was measured from the absorbance reading at 600 nm wavelength using the following formula: % Fragmented DNA = [OD(S)/[OD(S) + OD(P)] × 100 (OD: optical density, S: supernatant, P: pellet).

Statistical analysis
All data were analyzed using the General Liner Models (GLM) procedure of Statistical Analysis System (1982) [48] followed by Scheffé-test to assess significant differences between groups.The values are expressed as mean ± SEM.All statements of significance were based on the probability of P < 0.05.

Chemistry
The preparation of the targeted pyrrolopyrimidine derivatives 3-19 was started with the synthesis of cyclic pyrrole derivatives 2, which were prepared in three steps starting with acetophenone derivatives 1 as reported in the previous works of literature [20,33,49].The pyrrolo [2,3-d]pyrimidin-4-ones 3 or 6 were prepared via the reaction of 2 with formic acid to give pyrrolopyrimidine derivatives 3a and 3b, while compound 6 was obtained via the reaction of 2 with the mixture of acetic acid and hydrochloric acid in molar ratio 3:1 (Scheme 1).
The chlorination of pyrrolo [2,3-d]pyrimidin-4-one 3a was carried out in two ways.Refluxing of 3a in an excess of POCl 3 for around 18 h afforded 5 in moderate yield 72% in which the chlorination occurred on carbon-4 only.On the other hand, the microwave technique (MW) was used in the chlorination of 3a under the reaction condition of the presence of POCl 3 /P 2 O 5 for 15 min at 100 °C to afford 4a in which the chlorination occurred on the carbons 4 and 6 in excellent yield 92%. From these results we observed that microwave irradiation reduced the reaction time of the chlorination.Moreover, the heating using the microwave is more efficient for producing pure compounds in good to excellent yields in comparison to the refluxing conditions.Therefore, we have chlorinated both of the pyrrolo[2,3-d]pyrimidin-4-ones 3b and 6 using the microwave technique to afford the newly synthesized 4b and 7, respectively, as shown in Scheme 1.
Patrice Vanelle and co-workers reported that the insertion of the trichloromethyl group in position 2 of the quinazoline derivatives increased the antiplasmodial activity of these compounds [50][51][52].To study the structure-activity relationship (SAR), we prepared a new series of pyrrolo [2,3-d]pyrimidine derivatives with different substitutions on carbon 4 and chlorine on carbon 6 with or without trichloromethyl group at carbon 2 as shown in Scheme 2.Moreover, in the current work, we reported the first study for using these compounds as anti-cancer agents.The reaction of 4 and 7 with sodium methoxide in methanol afforded 8 with the trichloromethyl group in position 2, methoxy group in position 4 and chlorine atom in position 2, while in 9 the methoxy group in position 4 and chlorine atom in position 2. Likewise, the chlorine atom in position 4 of compounds 4 and 7 was replaced with pyrrolidine rings in 10 and 11 by refluxing the pyrrolidine with 4 or 7 in ethanol, respectively (Scheme 2).The treatment of 4a with thiourea in refluxing ethanol afforded 12 which reacted with MeI in ethanol and the presence of NaOH to give 13.Hydrazine monohydrate reacted with both 4 and 7 in ethanol to afford the corresponding pyrrolo [2,3-d]pyrimidine derivatives 14 and 15 as depicted in Scheme 2.
Likewise, the treatment of 4 or 7 with N-methyl piperazine or morpholine afforded a novel series of pyrrolopyrimidine derivatives having chlorine atoms at carbon 6 in compounds 16 and 18 (Scheme 3) and pyrrolo [2,3-d]

3-19 depicted absorption bands of the newly formed
C=N bond in the range of υ 1579-1642 cm −1 .The NH and NH 2 groups were assigned at υ 2998-3153 cm −1 and in the region of υ 3319-3322 cm −1 , and υ 3386-3416 cm −1 for the compounds containing NH and NH 2 , respectively.The 1 H-and 13 C-NMR spectra of compound 4 confirmed the chlorination of the carbons at positions 4 and 6 as well as the chlorination of the methyl group at position 2 of compound 6 using the MW technique, while the chlorination occurs only on carbon 4 using the reflux condition as shown in 5.All the protons and carbons of compounds 2-19 were assigned in their expected chemical shifts δ (ppm) as depicted in the experimental section.
Exclusively, good quality single crystals of 4b were obtained from a saturated solution of acetonitrile solution at room temperature as yellow crystals and found suitable for X-ray single crystal diffraction measurement (Fig. 2).Compound 4b crystallizes in the monoclinic space group C 2/c (see supplementary data   2) (for more details see Additional file 1).

Anti-cancer activity
All the synthesized compounds 3-19 were evaluated against selected human cancer cell lines for their antiproliferative activity in an in vitro study.Seven cancer cell lines MCF7, A549, HCT116, PC3, HePG2, PACA2 and the normal cell line (BJ1) were used in this study to evaluate the cytotoxicity of the compounds 3-19 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Doxorubicin (Dox.) was used as a reference standard drug.

Secondary screen
As illustrated in Table 3, the results indicated that most of the tested compounds have a promising cytotoxic effect against the tested cancer cell lines (MCF7, A549,

Molecular docking study
Compounds 14a and 17 were chosen for further molecular studies, as they had the most promising cytotoxic effect against MCF7 and PACA2 respectively.Herein, molecular docking studies were done to demonstrate the binding affinity of compounds 14a and 17 toward P53 mutant Y220C and anti-apoptotic Bcl2.Root mean squared deviation (RMSD) for P53 mutant Y220C and Bcl2 (PDB ID: 5O1H and 6QGG respectively) were 0.7 and 2.5 respectively indicating the high accuracy of docking results.The binding affinities of compound 14a toward P53 mutant Y220C and Bcl2 were − 14.98 and − 20.3 kcal/mole respectively which were very comparable to the standard value (− 15.82 and − 33.96 kcal/mole).Regarding compound 17, the binding energies were − 16 and − 22.8 kcal/mole toward P53 mutant Y220C and Bcl2 respectively as compared to the standard (− 15.82 and − 33.9 kcal/mole).As shown in Fig. 3a, compound 14a reactivated and stabilized p53 mutant Y220C through five interactions.One pi-anion hydrophobic interaction was seen between the benzene ring of dichlorobenzene moiety and ASP 228 with a bond distance of 5.61 A 0 .The remaining four interactions were pi-alkyl hydrophobic and the interacted amino acid residues were PRO 222 and PRO 223.It was noticed that compound 14a inhibited Bcl2 via eleven interactions.These interactions included; a hydrogen bond between N of pyrimidine ring and ARG 146 with a bond distance of 4.36 A 0 , a hydrogen bond between H of NH 2 moiety and GLU 136 (3.9A 0 ), attractive charge between the positive charge of N of NH 2 moiety and GLU 136, pi-cation interaction between benzene ring of 2,4-dichloro benzene and ARG 146, and the remaining seven interactions were pi-alkyl and alkyl interactions with (PHE 104, TYR 108, MET 115, LEU 137, and ALA 149) amino acid residues (Fig. 3b).Compound 17 interacted with P53 mutant Y220C through 17 interactions (Fig. 3c).Four hydrogen bonds between hydrogen of 1,4diazinane and (SER 227 and ASP 228) with bond distances 5.62, 4.83, 3.94 and 4.44 A 0 respectively, halogen interaction between Cl of dichlorobenzene moiety and ASP 228, and eleven alkyl and pi-alkyl hydrophobic interactions with VAL 147, PRO 151, CYS 220, PRO 222 and PRO 223 amino acid residues.At last, Fig. 3d showed the binding model of compound 17 with the active site of Bcl2 as followed: a carbon hydrogen bond between hydrogen of 1,4diazinane and ASP 111 (5.03A 0 ), pi-alkyl hydrophobic interaction between Br of 4-bromo benzene ring and (MET 115 and PHE 104), pialkyl between benzene of 4-bromobenzene moiety and MET 115, and pi-alkyl between Cl of pyrimidine ring and MET 115.So, from the above results, we could assume the activating effect of compounds 14a and 17 on P53 mutant Y220C and the inhibitory effect of compounds 14a and 17 against Bcl2 anti-apoptotic protein and this assumption was confirmed in the subsequent Eliza assay section.

Gene expression assay Effects on mRNA expression of P53, BAX, BCL2 on MCF7
The administration of 14a, 14b, 16b, 18b and Dox.up-regulated the mRNA levels of both P53 and BAX, whereas down-regulated Bcl-2 when compared with negative control.Moreover, we found that there are significant differences between all the compounds 14a, 14b, 16b, 18b and Dox. (Fig. 4).However, the injection of the newly synthesized compounds (14a, 14b, 16b and 18b) and Dox.down-regulated mRNA level of Bcl-2 when compared with negative control, there were also significant differences between 14a, 14b, 16b, 18b and Dox. (Fig. 4).

Effects on mRNA expression of CDK4, Il-8, DR4 and DR5 on MCF7
The treated MCF7 cell line with 14a, 14b, 16b, 18b and Dox.exhibited increased DR4 and DR5 mRNA levels, but these treatments lowered CDK4 and IL-8 mRNA when compared with negative control.In addition, there were significant differences between the administration of the newly synthesized pyrrolo [2,3-d] [55,56].In addition, TRAIL death receptors are known to be transcriptionally up-regulated by p53.[57,58] P53 has been shown to inhibit Bcl-2 activity and activated BAX and according to our data which revealed that compounds 14a, 14b, 16b and 18b significantly increased p53 mRNA levels,  5 The RT-qPCR validation of mRNA expression for P53, BAX, BcL2, CDK4, caspase-3, Il-8, DR4 (TRAIL1), and DR5 (TRAIL2), in, Paca2 cells among groups of control, (Dox.; negative control).Error bars represent the standard error of the mean (SEM).Means comparisons were performed by using the One-Way ANOVA test this could allow the upregulation of BAX, which in turn could inhibit IL-8 signaling and induce TRAIL death receptors expression.

Effects on mRNA expression of P53, BAX, BCL2 on Paca2
Compound 17 administration up-regulated the mRNA levels of both P53 and BAX in Paca2 cells, whereas it down-regulated Bcl-2 when compared to both Dox. and negative controls.Moreover, we found that there was no significant difference between negative control and Dox. (Fig. 5).

Effects on mRNA expression of CDK4, Casp-3, Il-8, DR4 and DR5 on Paca2
The treated Paca2 cell lines with 17 exhibited up-regulated Casp-3, DR4 and DR5 mRNA levels compared to both Dox. and negative control, while the treatment with 17 lowered CDK4 and IL-8 mRNA in comparison with both Dox. and negative control.In addition, there was no significant difference between administrations of Dox. and negative control (Fig. 5).

Effects on mRNA expression of P53, BAX, BCL2 on A549
The administration of 4b and Dox.up-regulated the mRNA levels of both P53 and BAX in A549 cells, whereas down-regulated Bcl-2 when compared with negative control.In addition, we found that there were significant differences between 4b and Dox. (Fig. 6).However, the injection of the 4b and Dox.down-regulated mRNA level of Bcl-2 when compared with negative control and also there were significant differences between 4b and Dox. (Fig. 6).

Effects on mRNA expression of CDK4, Casp-3, Il-8, DR4 and DR5 on A549
The treated lung cancer cells (A549) with 4b and the positive control (Dox.)showed up-regulated Casp-3, DR4 and DR5 mRNA levels.Moreover, it was observed considerable differences between the administration of 4b and Dox., while 4b is highly up-regulated of Casp-3, DR4 and DR5 mRNA levels and down-regulated CDK4 and IL-8 mRNA levels compared with the Dox.(Fig. 6).
Recently it was confirmed that the inhibition both of CDK4 and CDK6 genes influenced a varied range of cellular performances such as cancer cell metabolism and antitumor immunity.According to Shom et al. (2022) [59], Cyclin-dependent kinase (CDK4 and CDK6) are important mediators of cellular transference into the S phase and they are important growth and survival of many cancer types.According to our results, we found that both the newly synthesized pyrrolo [2,3-d]pyrimidine derivatives and Dox. were able to down-regulate CDK4 mRNA levels in treated cancer cells when compared with untreated cells (negative control) cells.In addition, the newly synthesized pyrrolo [2,3-d]pyrimidine derivatives were more potent in decreasing the level of mRNA of CDK4 than Dox.drug.6 The RT-qPCR validation of mRNA expression for P53, BAX, BcL2, CDK4, caspase-3, Il-8, DR4 (TRAIL1), and DR5 (TRAIL2), in, A549 cells among groups of control, (Dox., negative control).Error bars represent the standard error of the mean (SEM).Means comparisons were performed by using the One-Way ANOVA test

Eliza analysis
The activity of caspase 8, BAX, and Bcl2 in MCF7 cells after the treatment with the IC 50 of 14a, 14b, 16b and 18b was assessed using the ELIZA assay (Fig. 7).Also, the activity of caspase 3, caspase 8, BAX, and Bcl2 was determined in 17-treated Paca2 cells and 4b-treated A549 cells (Fig. 8).Regarding MCF7 cells, it was found that both compounds 14b and 16b significantly increased the expression level of caspase 8 and BAX, (18.263 and 14.72 pg/ml for caspase 8, respectively) (14.25 and 13.25 pg/ml for BAX, respectively) relative to the control (3.99 and 4.92 pg/ml, respectively).14a and 18b moderately enhanced the activity of caspase 8 and BAX, (8.76 and 10.29 pg/ml for caspase 8, respectively) (9.99 and 7.25 pg/ml for BAX, respectively).It was noticed that 14a and 16b greatly lowered the expression level of Bcl2 (2.4 and 4.25 pg/ml, respectively) relative to the control cells (14.37 pg/ml).The other two compounds 14b and 18b had a comparable effect on the activity of Bcl2 (8.25 and 9.24 pg/ml, respectively).As regard Paca2 cells, the activity of caspase 3, caspase 8 and BAX was significantly increased in response to 17 (9.14, 13.86 and 11.85 pg/ ml, respectively) as compared with control (5.34, 4.85 and 3.86 pg/ml, respectively).While the concentration of Bcl2 was greatly decreased by 17 (6.26pg/ml) compared to the control (16.23 pg/ml).In A549 cells, 4b increased the expression level of caspase 3, caspase 8 and BAX (12.12, 16.84 and 14.83 pg/ml, respectively) relative to the control (6.32, 5.83 and 5.84 pg/ml, respectively).While the expression level of Bcl2 was decreased (9.4 pg/ ml) compared to the control (16.21 pg/ml).Apoptosis is a programmed cell death and its regulation prevents many diseases including cancer.Caspases are a group of cysteine proteases that play a crucial role in apoptosis [60].There are two pathways of apoptosis, extrinsic (death receptor) and intrinsic (mitochondrial) pathways [60].In the extrinsic pathway, the activation of caspase 8 triggers the activation of executioner caspase 3 which leads to apoptosis [61].While in the intrinsic pathway, caspase 8 activates Bid and the remaining reactions occur in mitochondria [62].The mitochondrial pathway is regulated through Bcl2 family proteins, pro-apoptotic members such as BAX and anti-apoptotic members such as Bcl2 [62].So, the up-regulation of caspase 3, caspase 8 and BAX and down-regulation of Bcl2 as indicated in the above results demonstrated the induction of apoptosis in MCF7, Paca2 and A549 treated cells.

Flow cytometric analysis of cell cycle and apoptosis
We examined the cell cycle distribution after 24-h treatment of MCF7 with IC 50 concentrations of both the newly synthesized pyrrolopyrimidine compounds 14a and 14b to investigate the inhibitory effects on the proliferation of MCF7 cells.The untreated cells were used as a negative control for comparison purposes.As shown in Fig. 9, the percentage of cells in the G0/G1 phase was 11.43% for control cells, 68.60% for 14a treated cells, and 85.98% for 14b treated cells.It was noticed that the percentage of cells in the S phase was 1.91% for control cells, 17.61% for 14a treated cells, and 7.34% for 14b treated cells.While, the percentage of MCF7 cells in the G2/M phase was 72.58% in the control untreated cells, 4.93% in the 14a-treated cells, and 1.937% in the 14b-treated cells.So, compounds 14a and 14b caused cell cycle arrest at G1 and S phases as compared with the untreated control MCF7 cells.It was also notable that the percentage of treated arrested cells in the G1 phase was more than in the S phase and this coincided with our results in the gene expression section.As it was mentioned that both 14a and 14b down-regulated CDK4 which was responsible for G1/S phase progression.
Additionally, the percentage of apoptotic and necrotic cells in treated MCF7 and Paca2 cells was determined using the Annexin V-FITC/PI Double Staining Kit.After 24 h of treatment of MCF7 with IC 50 of 16b and 18b, it was found that the percentage of early apoptotic cells was increased to 11.49% and 20.63% respectively compared with untreated control cells (7.8%) (Fig. 10A).The percentage of late apoptotic cells was raised to 1.7% and 0.89% for 16b and 18b treated MCF7 cells respectively.While the percentage of necrotic cells was lowered to 0.96% and 0.76% respectively as compared with the control cells (1.01%).Regarding Paca2 cells, after 24 h of treatment with IC 50 of 17, it was found that the percentage of early and late apoptotic cells increased to 4.74% and 0.86% respectively.Also, the percentage of necrotic cells was raised to 6.17% compared with the control cells (2.27%) (Fig. 10B).

DNA fragmentation DNA fragmentation in Paca2
Determination of the rate of DNA fragmentation in the pancreatic cell line (Paca2) is depicted in Figs.11A and  12A.The results showed that the negative samples of Paca2 exhibited a significant decrease (P < 0.01) in DNA fragmentation values compared with those in the treated samples (17 and Dox.treated paca2 cells).However, the DNA fragmentation rates increased significantly (P < 0.01) in the treated Paca2 samples compared with the negative control.Moreover, the highest rate of DNA fragmentation was observed in Paca2-17 more than that found in the Dox.treated cells.

DNA fragmentation in A549
Determination of the value of DNA fragmentation in the lung cell line (A549) is illustrated in Figs.11B and 12B.The results revealed that the negative samples of A549 displayed a significant decrease (P < 0.01) in DNA fragmentation values compared with those in 4b treated samples and positive cancer cell line.However, the DNA fragmentation value was increased significantly (P < 0.01) in the treated A549-4b cancer cell line sample compared with the negative control.Moreover, the highest value of DNA fragmentation was observed in the positive cancer cell line more than those in the A549-4b cell line.

DNA fragmentation in MCF7
Assessment values of DNA fragmentation in MCF7 are summarized in Figs. 13 and 14.The results found that the negative samples of MCF7 showed a significant decrease (P < 0.01) in DNA fragmentation values compared with that observed in the treated samples (MCF7-14a, MCF7-14b, MCF7-16b and MCF7-18b) and positive cancer cell line.Conversely, the DNA fragmentation rates were found to be increased significantly (P < 0.01) in the treated MCF7 samples compared with the negative control.Moreover, the highest rate of DNA fragmentation was observed in MCF7-14a > MCF7-18b > MCF7-Positive > MCF7-16b > MCF7-14b cell line.

Conclusion
In summary, we reported a new series of pyrrolo [2,3-d] pyrimidine derivatives using the microwave technique as an eco-friendly method.The prepared compounds were evaluated in vitro as anti-cancer agents against several human cancer cell lines.The presence of trichloromethyl Where, most newly synthesized compounds might be considered as potent anticancer candidates for their ability to enhance the expression level of apoptotic genes (P53, BAX, DR4, DR5, and caspase-3), lower the expression of anti-apoptotic genes (CDK4, Bcl2, and Il-8), arrest the cell cycle at G1/S phase, induce apoptosis and cause DNA fragmentation in the selected cancer cell lines.

Fig. 3
Fig. 3 The molecular binding of compound 14a with the active site of a P53 and b Bcl2, and compound 17 with the active site of c P53 and d Bcl2

Fig. 7
Fig.7 The protein expression level of caspase 3, caspase 8, BAX, and Bcl2 for 14a, 14b, 16b and 18b treated MCF7 cells.The untreated MCF7 cells were used as a negative control.Data demonstrated the mean ± SE

17 Fig. 8 Fig. 9
Fig.8 The protein expression level of caspase 3, caspase 8, BAX, and Bcl2 for 17 treated Paca2 cells (left) and 4b treated A549 cells (right).The untreated cells were used as a negative control.Data represented mean ± SE

Fig. 10 Fig. 11 AFig. 12 A
Fig. 10 Flowcytometric analysis of apoptosis after 24 h of treatment for A MCF7 cells with IC 50 of 16b and 18b; B Paca2 cells with IC 50 of 17. Untreated cells were used as a negative control

Fig. 13 Fig. 14
Fig. 13 DNA fragmentation detected in breast cell line (MCF7) treated with 14a, 14b, 16b, and 18b.Means with different superscripts ( a, b, c, d, e ) between treatments in the same column are significantly different at P < 0.05

Table 1
Primers sequences

Table 2
MTT cell proliferation mortality assay (%) of cancer and normal cell lines at 100 μg/ml